Delivery of an agent often requires a carrier to get an active agent to target cells. Therapeutic effects in individuals are tied to those in compliance, and therefore oral delivery therapeutics tend to be more effective than injected therapeutics simply owing to better patient compliance with a dosing regime. The formulation of an oral agent is made quite difficult by oral active agent having to traverse strong stomach acid and digestive enzymes found within the gastrointestinal tract. After traversing these impediments, the oral active ingredient then must transport across cellular lipid membranes. While enteric coatings have been used successfully in protecting active agents from stomach acid and enzyme inhibiting adjuvants are suitable to prevent enzymatic degradation, enhancing bioavailability through transport of an active agent across epithelial cell membranes has proven more difficult. Traditional approaches to enhance epithelial cell permeability to an active agent have included compounding with adjuvants such as resorinol, surfactants, polyethylene glycol or bioacids. Alternatively, microencapsulation with liposomes, polysaccharides have also been used to limit enzymatic degradation as well as enhanced permeability.
More recently, active transport of active agents has been investigated to enhance bioavailability of otherwise promising active agents that alone have insufficient movement across epithelial cell membranes, resulting in dosing regimes causing significant side effects. Peptide transport is a specific biochemical process in which small peptides are transported across a membrane by energy dependent saturatable carriers. PEPT1 and PEPT2 are known transporters found in intestinal and renal absorptive epithelial cells.
With an appreciation that peptide transporters are found in intestinal and renal absorptive epithelial cells, attempts have been made to conjugate active agents to peptides, in order to carry active agents through epithelial cell membranes. These efforts have principally focused on coupling an active agent to a C-terminus of a dipeptide in order to enhance affinity. Efforts to attach a dye to the C-terminus of a dipeptide including lysine showed great affinity yet no transport. Abe H. et al., Bioconjug. Chem. 1999; 10:24–31. Other attempts to gain transport of active agents with a dipeptide have involved the use of a D-amino acid coupled to an L-amino acid that in turn is conjugated to a species of interest. Groneberg D. A. et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281:G697–704. Additionally, a β-amino acid N-terminus has been used to couple to L-lysine which in turn is conjugated to a marker of interest. Otto C. et al., Anat. Rec. 1996; 245:662–7. As a whole, these attempts have met with limited acceptance as a viable active agent carrier system.
In addition to in vivo delivery, a variety of cell culture based systems have been developed to screen for compounds, including the human intestinal cell lines, Caco-2 and HT-29 cells and Madin-Darby canine kidney (MDCK) cells. U.S. Pat. No. 5,856,189 and Walter et al., Journal of Pharmaceutical Sciences, 1996; 85(10):1070 and Veronesi B., Neurotoxicology, 1996 Summer; 17(2):433–43. These cell lines have proven useful to assess permeability of passively absorbed compounds, which do not require a specific transporter for absorption. While these cells do express certain transporters, the expression of these transporters varies with age of cells and growth conditions and consequently these cells are not suitable for testing drugs that target specific transporters.
There are cell-based systems in which specific transporters, including the ileal/renal bile acid transporter and the human renal organic anion transporter 1 (hOAT1), have been expressed and are used to screen for modulators of transport activity with the aim of control of blood cholesterol levels, treatments of diabetes, heart, liver, certain digestive disorders, and assessment of nephrotoxic and nephroprotectant effects as described in U.S. Pat. Nos. 5,869,265 and 6,479,231.
Cultured cell lines of intestinal and renal origin that express PEPT1 and PEPT2 have been identified. (Dantzig et el., Science 1994; 264:430–433; Liang et al., J. Biol. Chem. 1995; 270:6456–6463 and Oh et al., Pharm. Biotechnol. 1999; 12:59–88). With these cultured cell lines, the assessment of short peptide carrier systems for delivery of agents to epithelial cells has been greatly enhanced.
In spite of the existence of cultured cell lines expressing various transporters and chemistries for conjugating an active agent to a short peptide carrier, there still exists a need for an effective short peptide carrier system for cellular delivery of active agents.